Engineering m⁶A demethylation-activated DNAzyme for visually and sensitively sensing fat mass and obesity-associated...

Fat mass and obesity-associated protein (FTO) regulating the N6-methyladenine (m⁶A, the most pervasive epigenetic modification) levels within the nucleus has been identified as a potential biomarker for cancer diagnosis and prognosis. However, current methods for FTO detection are complicated or/and not sensitive enough for practical application. Herein, we propose a colorimetric biosensor for detecting FTO based on a delicate design of m⁶A demethylation-activated DNAzyme. Specifically, an m⁶A-blocked DNAzyme is constructed as a switch of the biosensor that can be turned on by target FTO. The decreased thermal stability resulting from substrate cleavage leads to a DNAzyme recycling to produce multiple primers. Then the rolling circle amplification (RCA) reactions can be initiated to generate G-quadruplex-DNAzymes catalyzing 2,2-azino-bis-(3-ethylben-zthiazoline-6-sulfonic acid (ABTS) oxidation which can be readily observed by the naked eye. Quantitative detection can also be achieved with a limit of detection (LOD) down to 69.9 fM, exhibiting higher sensitivity than previous reports. Therefore, this biosensor opens a simple and sensitive way to achieve visual assay of FTO via triple signal amplification. In addition, our biosensor has been successfully applied to FTO detection in clinical samples, which shows great potential in clinical molecular diagnostics.